Prof Peter Chantler
Molecular Biology of the Cell
c. Creation of a DNA library
The next step involves inserting a complete set of representative DNA fragments into cloning vectors, a process known as creating a library.
Construction of a genomic library
The simplest example is that of genomic library construction - all you have to do is chop up total DNA extracted from somatic cells with restriction enzymes. The restriction enzymes should be chosen so that, on average, they will create DNA fragments of appropriate size to be inserted into each vector molecule.
Typically, one does not use an enzyme that cuts frequently (eg. a 4-base cutter) because the fragments generated will be too short; equally, one does not use a rare base cutter as this will generate a library with a small selection of inserts, each too large to be useful.
Construction of a cDNA library
Construction of a cDNA library is more complicated because one first has to synthesize the cDNA. First, mRNA is prepared (characterized by a long poly-A tail at the 3' end, so it can be extracted using an oligo-dT column). Following this, oligo-dT primers are then annealed to the poly-A tail, which facilitates synthesis of a DNA copy through the use of reverse transcriptase. The original RNA strand is then removed by alkaline hydrolysis. DNA polymerase is used to synthesize the second DNA strand and this is initiated at various sites through the use of random hexamer nucleotide primers. Use of a DNA ligase covalently seals the remaining breaks.
The cDNA may be inserted into a phage vector, such as lgt10, in order to create a library. Phage DNA is readied for this process by cleavage with EcoRI. The cDNA that is to be inserted into the phage is first treated with EcoRI methylase so as to protect EcoRI sites in the cDNA from subsequent cleavage.
The blunt-ended cDNA is made compatible with the EcoRI sites through joining on synthetic linkers that contain EcoRI sites; these are joined covalently through the action of DNA ligase. Upon treatment with EcoRI, which creates a single sticky site at either end of the cDNA, the insert and the cleaved l phage can be annealed. Note that because adjacent phage can also join together via their cos sites, concatamers result. These are broken down into amounts of DNA required to create single phage genomes during the packaging of this DNA to form viable phage particles. These phage particles can be propagated through plaque formation on a bacterial lawn.
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Course updated 6 Apr 2004